Abstract Body (Do not enter title and authors here): Background information: Hypertrophic cardiomyopathy (HCM) is the commonest inherited cardiovascular disease. Whilst important for family screening, the utility of genetic testing is limited by a persistent low yield. This may be partly explained by only exonic and canonical splice site variants considered clinically significant, while intronic variation is overlooked. Mutations in MYBPC3 are a common cause of HCM, but studies into the pathogenesis of MYBPC3-HCM are hindered by the need for myocardial tissue. Our previous work has shown that intronic MYBPC3 variants disrupt splicing in patient derived iPSC-CMs, suggesting a role for intronic MYBPC3 variation in HCM pathogenesis.
Aim: Herein we sought to characterise iPSC-CMs generated from patients harbouring deep intronic MYBPC3 variants (c.1224-52G>A (-52) & c.1898-23A>G (-23)) and establish any morphological and physiological abnormalities arising from splicing alterations consistent with the HCM phenotype.
Methods: Patient-derived iPSC-CMs were characterised for cellular features suggestive of HCM by analysis of contractility, calcium handing ability, western blot of MYBPC3 protein levels, and immunocytochemistry to examine cellular morphology and sarcomeric arrangement.
Results: Upon differentiation to cardiomyocytes, iPSC-CMs from both -52 and -23 variants exhibited splicing abnormalities, albeit through different mechanisms. The -52 variant produced mutant mRNA that was degraded by regulatory mechanisms. The -23 also produced aberrant mRNA transcripts but were shown to escape such degradation. Both variants showed abnormalities in contractile ability and calcium handling compared to a wildtype (WT). Cells of both variants also showed morphological features consistent with HCM of sarcomeric disarray, large cell size, cell roundness, and multinucleation. Western blot analysis showed lower overall MYBPC3 protein levels for both variants compared to a WT, agreeing well with current literature that haploinsufficiency underpins the pathogenicity of MYBPC3 mutations in HCM.
Conclusion: Functional analysis of iPSC-CMs harbouring deep intronic MYBPC3 variants exhibit splicing abnormalities alongside structural and physiological impairments consistent with the HCM phenotype. These findings add to growing evidence that intronic variation may play a role in HCM pathogenesis and highlights the importance of expanding genetic testing beyond traditional regions in gene-elusive individuals.